Signaling pathways between the extracellular environment and the nucleus of a cell involve the formation of many molecular complexes in which multiple proteins are assembled to directly or indirectly induce molecular events, such as enzyme activation or de-activation, Gomperts et al, Signal Transduction (Academic Press, N.Y., 2002). Such pathways and their components have been the subject of intense investigation because of the role aberrant pathway behavior plays in many disease conditions, especially cancer, e.g. McCormick, Trends in Cell Biology, 9: 53-56 (1999); Blume-Jensen and Hunter, Nature, 411: 355-365 (2001); Nicholson et al, Cellular Signalling, 14: 381-395 (2002); and the like. It has been observed that many cancers are associated with an accumulation of mutations or other genetic alterations that affect components of signaling pathways (e.g. by over expression), particularly those pathways involved with cell proliferation, cell motility, differentiation, and cell death, e.g. Blume-Jensen and Hunter (cited above).
Receptor tyrosine kinases are an important class of receptor that are involved in many fundamental cellular processes including cell proliferation, survival, metabolism, and migration, e.g. Schlessinger, Cell, 103: 211-225 (2000). Prominent families of this class include epidermal growth factor receptor (EGFR or Her1), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endotheilial growth factor receptor (VEGFR), Blume-Jensen and Hunter (cited above). The class of receptor tyrosine kinases is so named because when activated by dimerization, the intracellular domain of RTKs acquire tyrosine kinase activity that can, in turn, activate a variety signal transduction pathways. Because of this property, many receptor tyrosine kinases have been used as targets for drug development, and several promising clinical-phase drugs, such as Iressa and Tarceva, have been designed to inhibit RTK activity, e.g. Taxler, Expert Opin. Ther. Targets, 7: 215-234 (2003). The availability of convenient methods for measuring pathway activation would lead to better understanding of drug mechanisms, and to better drug selection and disease management.
Signaling pathways have been difficult to study not only because of their complexity and interconnectedness, but also because of the disruptive procedures required for analysis of intracellular complexes, e.g. Weng et al, Science, 284: 92-96 (1999); Machida et al, Molecular & Cellular Proteomics, 2.4: 215-233 (2003); Price et al, Methods in Molecular Biology, 218: 255-267 (2003). A wide variety of techniques have been used to study cellular protein-protein interactions and complexes, including immunoprecipitation, chemical cross-linking, yeast two-hybrid systems, tagged fusion proteins, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), mass spectroscopy, and the like, e.g. Golemis, editor, Protein-Protein Interactions (Cold Spring Harbor Laboratory Press, New York, 2002); Price et al (cited above); Sorkin et al, Curr. Biol., 10: 1395-1398 (2000); McVey et al, J. Biol. Chem., 17: 14092-14099 (2001); Salim et al, J. Biol. Chem., 277: 15482-15485 (2002); Angers et al, Proc. Natl. Acad. Sci., 97: 3684-3689 (2000); Jones et al, Proteomics, 2: 76-84 (2002); and Petricoin III, et al, The Lancet, 359: 572-577 (2002). Unfortunately, such techniques are difficult to apply, generally lack sufficient sensitivity to provide an accurate picture of the state of a signaling pathway, and/or cannot measure multiple components or interacting components that are crucial for pathway activation.
In view of the above, the availability of a convenient and sensitive assay for determining activation status of RTK signaling pathways, particularly signaling pathways activated by ErbB receptors, would lead to significant medical advances, especially in the area of cancer treatment and drug development.